Apilimod compositions and methods for using same in the treatment of renal cancer

ABSTRACT

The present disclosure relates to methods for treating renal cancer with apilimod and related compositions and methods.

RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.16/226,175, filed Dec. 19, 2018, now abandoned, which is a continuationof U.S. Ser. No. 15/524,884, filed May 5, 2017, now U.S. Pat. No.10,206,910 issued Feb. 19, 2019, which is a national stage entry, filedunder 35 U.S.C. § 371, of International Application No.PCT/US2015/059512, filed on Nov. 6, 2015, which claims priority to U.S.Ser. No. 62/077,127, filed on Nov. 7, 2014, the contents of which arehereby fully incorporated by reference.

FIELD OF THE DISCLOSURE

The present disclosure relates to compositions comprising apilimod andmethods of using same for the treatment of renal cancer.

BACKGROUND OF THE DISCLOSURE

Apilimod, also referred to as STA-5326, hereinafter “apilimod”, isrecognized as a potent transcriptional inhibitor of IL-12 and IL-23. Seee.g., Wada et al. Blood 109 (2007): 1156-1164. IL-12 and IL-23 areinflammatory cytokines normally produced by immune cells, such asB-cells and macrophages, in response to antigenic stimulation.Autoimmune disorders and other disorders characterized by chronicinflammation are characterized in part by inappropriate production ofthese cytokines. In immune cells, the selective inhibition ofIL-12/IL-23 transcription by apilimod was recently shown to be mediatedby apilimod's direct binding to phosphatidylinositol-3-phosphate5-kinase (PIKfyve). See, e.g., Cai et al. Chemistry and Biol. 20(2013):912-921. PIKfyve plays a role in Toll-like receptor signaling,which is important in innate immunity.

Based upon its activity as an immunomodulatory agent and a specificinhibitor of IL-12/IL-23, apilimod has been proposed as useful intreating autoimmune and inflammatory diseases and disorders. See e.g.,U.S. Pat. Nos. 6,858,606 and 6,660,733 (describing a family ofpyrimidine compounds, including apilimod, purportedly useful fortreating diseases and disorders characterized by IL-12 or IL-23overproduction, such as rheumatoid arthritis, sepsis, Crohn's disease,multiple sclerosis, psoriasis, or insulin dependent diabetes mellitus).Similarly, apilimod was suggested to be useful for treating certaincancers based upon its activity to inhibit c-Rel or IL-12/23,particularly in cancers where these cytokines were believed to play arole in promoting aberrant cell proliferation role. See e.g., WO2006/128129 and Baird et al., Frontiers in Oncology 3:1 (2013,respectively).

Each of three clinical trials of apilimod has focused on its potentialefficacy in autoimmune and inflammatory diseases. The trials wereconducted in patients having psoriasis, rheumatoid arthritis, andCrohn's disease. An open label clinical study in patients with psoriasisconcluded that oral administration of apilimod showed immunomodulatoryactivity supporting the inhibition of IL-12/IL-23 synthesis for thetreatment of TH1- and TH17-mediated inflammatory diseases. Wada et al.,PLosOne 7:e35069 (April 2012). But the results of controlled trials inrheumatoid arthritis and Crohn's disease did not support the notion thatIL-12/IL-23 inhibition by apilimod translates into clinical improvementin either of these indications. In a randomized, double-blind,placebo-controlled Phase II clinical trial of apilimod in patients withrheumatoid arthritis, apilimod failed to alter synovial IL-12 and IL-23expression. Krauz et al., Arthritis & Rheumatism 64:1750-1755 (2012).The authors concluded that the “results do not support the notion theIL-12/IL-23 inhibition by apilimod is able to induce robust clinicalimprovement in RA.” Similarly, a randomized, double-blind,placebo-controlled trial of apilimod for treatment of active Crohn'sdisease concluded that, although well tolerated, apilimod did notdemonstrate efficacy over placebo. Sands et al Inflamm Bowel Dis. 2010July; 16(7):1209-18.

The mammalian target of rapamycin (mTOR) pathway is an importantcellular signaling pathway that is involved in multiple physiologicalfunctions, including cell growth, cell proliferation, metabolism,protein synthesis, and autophagy (La Plante et al Cell 2012, (149 (2),pp. 274-293). mTOR is a kinase that integrates intracellular andextracellular cues that signal the levels of amino acids, stress,oxygen, energy, and growth factors and regulates the cellular responseto these environment cues. mTOR deregulation has been implicated in awide range of disorders and diseases, including cancer, obesity,diabetes, and neurodegeneration. Certain components of the mTOR pathwayhave been explored as drug targets for treating some of these diseases.However, therapeutic efficacy has been limited, for example, in thetreatment of some cancers, and some mTOR inhibitors have been shown tohave an adverse effect on metabolism. The tuberous sclerosis complextumor suppressor genes, TSC1 and TSC2, are negative regulators of mTOR.

SUMMARY OF THE DISCLOSURE

The present disclosure is based in part on the surprising discovery thatapilimod is a highly cytotoxic agent in TSC null cells. In these cells,the mTOR pathway is constitutively active. The mTOR pathway is activatedin a number of cancers, and in further screening of over 100 cancer celllines apilimod showed anti-proliferative activity in cell lines fromdiverse cancers, including renal cell carcinomas. Furthermore, thecytotoxic activity of apilimod in cancer cell lines was due to aninhibition of intracellular trafficking and a corresponding increase inapoptosis and/or autophagy, rather than via apilimod's inhibition ofIL-12/23 production, as would have been predicated based upon apilimod'simmunomodulatory activity. In addition, a screen over 450 kinasesidentified PIKfyve as the only high affinity binding target (Kd=75 pM)for apilimod. The present disclosure provides new methods for thetherapeutic use of apilimod in treating cancer renal cell cancer.

In one aspect, the disclosure provides a composition for treating renalcancer in a subject having renal cancer, the composition comprising atherapeutically effective amount of apilimod, or a pharmaceuticallyacceptable salt thereof. In embodiments, the apilimod is apilimoddimesylate. In embodiments, the composition is in a form suitable fororal or intravenous administration. In embodiments, the compositionfurther comprises at least one additional active agent, which may beselected from a therapeutic agent or a non-therapeutic agent, or acombination of a therapeutic agent and a non-therapeutic agent. Inembodiments, the at least one additional active agent is a therapeuticagent selected from the group consisting of a protein kinase inhibitor,a platinum based anti-neoplastic agent, a topoisomerase inhibitor, anucleoside metabolic inhibitor, an alkylating agent, an intercalatingagent, a tubulin binding agent, and combinations thereof. Inembodiments, the therapeutic agent is a protein kinase inhibitor. Inembodiments, the protein kinase inhibitor is pazopanib or sorafenib, ora combination thereof. The composition may further comprise anon-therapeutic agent selected to ameliorate one or more side effects ofthe apilimod. In embodiments, the non-therapeutic agent is selected fromthe group consisting of ondanestron, granisetron, dolsetron, andpalonosetron. In embodiments, the non-therapeutic agent is selected fromthe group consisting of pindolol and risperidone.

In embodiments, the composition comprises an amount of the apilimoddimesylate effective to inhibit PIKfyve kinase activity in the cancercells of the subject. The renal cancer may in some embodiments berefractory to standard treatment or metastatic. In embodiments, therenal cancer is selected from clear cell renal carcinoma, a transitionalcell carcinoma, Wilms tumor (nephroblastoma), renal sarcoma, and benign(non-cancerous) kidney tumors, renal adenoma, oncocytoma, andangiomyolipoma.

In one aspect, the disclosure provides a method for treating renalcancer in a subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of apilimod, or acomposition comprising apilimod, wherein the apilimod is apilimod itself(i.e., apilimod free base), or a pharmaceutically acceptable salt,solvate, clathrate, hydrate, polymorph, prodrug, analog or derivativethereof. In one embodiment, the apilimod is apilimod free base orapilimod dimesylate.

In embodiments, the method further comprises administering at least oneadditional active agent to the subject. The at least one additionalactive agent may be a therapeutic agent or a non-therapeutic agent. Theat least one additional active agent may be administered in a singledosage form with the apilimod, or in a separate dosage form from theapilimod. In embodiments, the at least one additional active agent is atherapeutic agent selected from the group consisting of a protein kinaseinhibitor, a platinum based anti-neoplastic agent, a topoisomeraseinhibitor, a nucleoside metabolic inhibitor, an alkylating agent, anintercalating agent, a tubulin binding agent, PD-1/PDL-1 pathwayinhibitor, and combinations thereof. In embodiments, the therapeuticagent is a protein kinase inhibitor. In embodiments, the protein kinaseinhibitor is pazopanib or sorafenib, or a combination thereof. Inembodiments, the at least one additional active agent is a therapeuticagent selected from the group consisting of sorafenib (Nexavar®),sunitinib (Sutent®) temsirolimus (Torisel®), everolimus (Afinitor®),bevacizumab (Avastin®), pazopanib (Votrient®), axitinib (Inlya®) andcombinations thereof. In embodiments, the therapeutic agent is aPD-1/PDL-1 pathway inhibitor. In embodiments, the PD-1/PDL-1 pathwayinhibitor is selected from pembrolizumab (Keytruda), avelumab,atezolizumab (MPDL3280A), nivolumab (BMS-936558), pidilizumab (MK-3475),MSB0010718C, and MEDI4736.

In embodiments, the at least one active agent is a non-therapeutic agentselected to ameliorate one or more side effects of apilimod. Inembodiments, the non-therapeutic agent is selected from the groupconsisting of ondanestron, granisetron, dolsetron, and palonosetron. Inone embodiment, the non-therapeutic agent is selected from the groupconsisting of pindolol and risperidone. In one embodiment, the dosageform of the apilimod composition is an oral dosage form. In anotherembodiment, the dosage form of the apilimod composition is suitable forintravenous administration, administration is by a single injection orby a drip bag.

In one embodiment, the subject is a human cancer patient. In oneembodiment, the human cancer patient in need of treatment with apilimodis on whose cancer is refractory to a standard chemotherapy regimen. Inone embodiment, the human cancer patient in need of the treatment withapilimod is one whose cancer as recurred following treatment with astandard chemotherapy regimen. In one embodiment, the cancer is a renalcancer. In one embodiment, the renal cancer is a transitional cellcarcinoma, Wilms tumor (nephroblastoma), renal sarcoma, and benign(non-cancerous) kidney tumors, renal adenoma, oncocytoma, andangiomyolipoma. In one embodiment, the renal cancer is a clear cellrenal cell carcinoma.

In one embodiment, the standard chemotherapy regimen comprises one ormore therapeutic agents selected from the group consisting of ibrutinib,rituximab, doxorubicin, prednisolone, vincristine, velcade,cycloposphoamide, dexamethasone and everolimus.

In one embodiment, the method is a method for treating renal cancerusing a combination therapy comprising apilimod and a chemotherapyregimen for the treatment of the renal cancer. In embodiments, thechemotherapy regimen comprises a PD-1/PDL-1 pathway inhibitor. Inembodiments, the PD-1/PDL-1 pathway inhibitor is selected frompembrolizumab (Keytruda), avelumab, atezolizumab (MPDL3280A), nivolumab(BMS-936558), pidilizumab (MK-3475), MSB0010718C, and MEDI4736.

In another embodiment, the method is a method for treating renal cancerusing a combination therapy comprising apilimod and an immunotherapyregimen for the treatment of the renal cancer. In one embodiment theimmunotherapy regime is the Interleukin-2 (IL-2) regime or thealpha-interferon regime. In one embodiment, the immunotherapy regimencomprises a PD-1/PDL-1 pathway inhibitor. In embodiments, the PD-1/PDL-1pathway inhibitor is selected from pembrolizumab (Keytruda), avelumab,atezolizumab (MPDL3280A), nivolumab (BMS-936558), pidilizumab (MK-3475),MSB0010718C, and MEDI4736.

In some embodiments, the method is a method for treating renal cancerusing a combination therapy comprising apilimod and a protein kinaseinhibitor regimen for the treatment of the renal cancer. In oneembodiment the protein kinase inhibitor regimen is sorafenib, sunitinib,bevacizumab, lenvatinib, everolimus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: TSC2 deficient cells are highly sensitive to apilimod (IC₅₀=20nM).

FIGS. 2A-B: A, percentage of cell lines with IC50 less than 500 nM; B,normal lung fibroblasts are not sensitive to apilimod-inducedcytotoxicity at apilimod concentrations as high as 10 micromolar.

FIG. 3: apilimod induces autophagy in a dose-dependent manner.

FIG. 4: Volcano plot of significant captured hits applying CT-689 at 0.1μM concentration under optimized capture conditions.

FIG. 5: apilimod binds with high affinity to PIKfyve (Kd=75 pM).

FIG. 6: antiproliferative activity of LAM-002 in renal clear cellcarcinoma cell line 769-P, Avg. IC₅₀=44 nM (n=2).

FIG. 7: antiproliferative activity of LAM-002 in renal clear cellcarcinoma cell line RCC-MF, Avg. IC₅₀=8 nM (n=2).

FIG. 8: antiproliferative activity of LAM-002 in renal clear cellcarcinoma cell line RCC-ER, Avg. IC₅₀=9 nM (n=2).

FIG. 9: antiproliferative activity of LAM-002 in renal clear cellcarcinoma cell line RCC-FG2, Avg. IC₅₀=32 nM (n=2).

FIG. 10: antiproliferative activity of LAM-002 in renal clear cellcarcinoma cell line RCC-JF, Avg. IC₅₀=60 nM (n=2).

FIG. 11: antiproliferative activity of LAM-002 in renal clear cellcarcinoma cell line 786-O, Avg. IC₅₀=71 nM (n=2).

FIG. 12: antiproliferative activity of LAM-002 in renal clear cellcarcinoma cell line A704, Avg. IC₅₀=11 nM (n=2).

FIG. 13: antiproliferative activity of LAM-002 in renal clear cellcarcinoma cell line RCC-JW, Avg. IC₅₀=27 nM (n=2).

FIGS. 14A-B: LAM-002+sorafenib combination in RCC-ER cells (5 dayassay). A, bar graph showing cell viability (%); B, determination of thecombination index (CI) value at ED₅₀, ED₇₅ and ED₉₀.

FIGS. 15A-B: LAM-002+sorafenib combination in RCC-FG2 cells (5 dayassay). A, bar graph showing cell viability (%); B, determination of thecombination index (CI) value at ED₅₀, ED₇₅ and ED₉₀.

FIGS. 16A-B: LAM-002+sorafenib combination in RCC-MF cells (5 dayassay). A, bar graph showing cell viability (%); B, determination of thecombination index (CI) value at ED₅₀, ED₇₅ and ED₉₀.

FIGS. 17A-B: LAM-002+sorafenib combination in 769-P cells (5 day assay).A, bar graph showing cell viability (%); B, determination of thecombination index (CI) value at ED₅₀, ED₇₅ and ED₉₀.

FIGS. 18A-B: LAM-002+pazopanib combination in RCC-ER cells (5 dayassay). A, bar graph showing cell viability (%); B, determination of thecombination index (CI) value at ED₅₀, ED₇₅ and ED₉₀.

FIGS. 19A-B: LAM-002+pazopanib combination in RCC-FG2 cells (5 dayassay). A, bar graph showing cell viability (%); B, determination of thecombination index (CI) value at ED₅₀, ED₇₅ and ED₉₀.

FIGS. 20A-B: LAM-002+pazopanib combination in RCC-MF cells (5 dayassay). A, bar graph showing cell viability (%); B, determination of thecombination index (CI) value at ED₅₀, ED₇₅ and ED₉₀.

FIGS. 21A-B: LAM-002+pazopanib combination in 769-P cells (5 day assay).A, bar graph showing cell viability (%); B, determination of thecombination index (CI) value at ED₅₀, ED₇₅ and ED₉₀.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure provides compositions and methods related to theuse of apilimod for treating renal cancer in a subject, preferably ahuman subject, in need of such treatment. The present disclosuregenerally relates to new uses of apilimod based upon the surprisingdiscovery of apilimod's cytotoxic activity against a range of cancercells of both lymphoid and non-lymphoid origin, an activity that is notclearly related to, or predictable from, apilimod's knownimmunomodulatory and IL-12/23 inhibitory activity.

In addition, the present disclosure provides novel therapeuticapproaches to cancer treatment based upon combination therapy utilizingapilimod and at least one additional therapeutic agent. The combinationtherapies described herein exploit the unique cytotoxic activity ofapilimod which is shown to provide a synergistic effect when combinedwith other anti-cancer agents.

As used herein, the term “apilimod” may refer to apilimod itself (i.e.,apilimod free base), or may encompass pharmaceutically acceptable salts,solvates, clathrates, hydrates, polymorphs, metabolites, prodrugs,analogs or derivatives of apilimod, as described below. The structure ofapilimod is shown in Formula I:

The chemical name of apilimod is2-[2-Pyridin-2-yl)-ethoxyl-4-N′-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine(IUPAC name:(E)-4-(6-(2-(3-methylbenzylidene)hydrazinyl)-2-(2-(pyridin-2-yl)ethoxy)pyrimidin-4-yl)morpholine),and the CAS number is 541550-19-0.

Apilimod can be prepared, for example, according to the methodsdescribed in U.S. Pat. Nos. 7,923,557, and 7,863,270, and WO2006/128129.

As used herein, the term “pharmaceutically acceptable salt,” is a saltformed from, for example, an acid and a basic group of apilimod.Illustrative salts include, but are not limited, to sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, besylate, gentisinate, fumarate, gluconate,glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(e.g., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

The term “pharmaceutically acceptable salt” also refers to a saltprepared from an apilimod composition having an acidic functional group,such as a carboxylic acid functional group, and a pharmaceuticallyacceptable inorganic or organic base.

The term “pharmaceutically acceptable salt” also refers to a saltprepared from apilimod having a basic functional group, such as an aminofunctional group, and a pharmaceutically acceptable inorganic or organicacid.

The salts of the compounds described herein can be synthesized from theparent compound by conventional chemical methods such as methodsdescribed in Pharmaceutical Salts: Properties, Selection, and Use, P.Hemrich Stalil (Editor), Camille G. Wermuth (Editor), ISBN:3-90639-026-8, August 2002. Generally, such salts can be prepared byreacting the parent compound (e.g.,2-[2-Pyridin-2-yl)-ethoxyl-4-N′-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine)with the appropriate acid in water or in an organic solvent, or in amixture of the two.

One salt form of a compound described herein can be converted to thefree base and optionally to another salt form by methods well known tothe skilled person. For example, the free base can be formed by passingthe salt solution through a column containing an amine stationary phase(e.g. a Strata-NH₂ column). Alternatively, a solution of the salt inwater can be treated with sodium bicarbonate to decompose the salt andprecipitate out the free base. The free base may then be combined withanother acid using routine methods.

As used herein, the term “polymorph” means solid crystalline forms of acompound of the present disclosure (e.g.,2-[2-Pyridin-2-yl)-ethoxyl-4-N′-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine)or complex thereof. Different polymorphs of the same compound canexhibit different physical, chemical and/or spectroscopic properties.Different physical properties include, but are not limited to stability(e.g., to heat or light), compressibility and density (important informulation and product manufacturing), and dissolution rates (which canaffect bioavailability). Differences in stability can result fromchanges in chemical reactivity (e.g., differential oxidation, such thata dosage form discolors more rapidly when comprised of one polymorphthan when comprised of another polymorph) or mechanical characteristics(e.g., tablets crumble on storage as a kinetically favored polymorphconverts to thermodynamically more stable polymorph) or both (e.g.,tablets of one polymorph are more susceptible to breakdown at highhumidity). Different physical properties of polymorphs can affect theirprocessing. For example, one polymorph might be more likely to formsolvates or might be more difficult to filter or wash free of impuritiesthan another due to, for example, the shape or size distribution ofparticles of it.

As used herein, the term “hydrate” means a compound of the presentdisclosure (e.g.,2-[2-Pyridin-2-yl)-ethoxyl-4-N′-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine)or a salt thereof, which further includes a stoichiometric ornon-stoichiometric amount of water bound by non-covalent intermolecularforces.

As used herein, the term “clathrate” means a compound of the presentdisclosure (e.g.,2-[2-Pyridin-2-yl)-ethoxyl-4-N′-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine)or a salt thereof in the form of a crystal lattice that contains spaces(e.g., channels) that have a guest molecule (e.g., a solvent or water)trapped within.

As used herein, the term “prodrug” means a derivative of a compounddescribed herein (e.g.,2-[2-Pyridin-2-yl)-ethoxyl-4-N′-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine)that can hydrolyze, oxidize, or otherwise react under biologicalconditions (in vitro or in vivo) to provide a compound of thedisclosure. Prodrugs may only become active upon such reaction underbiological conditions, or they may have activity in their unreactedforms. Examples of prodrugs contemplated in this disclosure include, butare not limited to, analogs or derivatives of a compound describedherein (e.g.,2-[2-Pyridin-2-yl)-ethoxyl-4-N′-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine)that comprise biohydrolyzable moieties such as biohydrolyzable amides,biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzablecarbonates, biohydrolyzable ureides, and biohydrolyzable phosphateanalogues. Other examples of prodrugs include derivatives of compoundsof any one of the formulae disclosed herein that comprise —NO, —NO₂,—ONO, or —ONO₂ moieties. Prodrugs can typically be prepared usingwell-known methods, such as those described by Burger's MedicinalChemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolffed., 5th ed).

As used herein, the term “solvate” or “pharmaceutically acceptablesolvate,” is a solvate formed from the association of one or moresolvent molecules to one of the compounds disclosed herein (e.g.,2-[2-Pyridin-2-yl)-ethoxyl-4-N′-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine).The term solvate includes hydrates (e.g., hemi-hydrate, mono-hydrate,dihydrate, trihydrate, tetrahydrate, and the like).

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound. As used herein,the term “derivative” refers to compounds that have a common corestructure, and are substituted with various groups as described herein.

Methods of Treatment

The present disclosure provides methods for the treatment of renalcancer in a subject in need thereof by administering to the subject atherapeutically effective amount of apilimod, or a pharmaceuticallyacceptable salt, solvate, clathrate, hydrate, polymorph, prodrug, analogor derivative thereof.

In one embodiment the renal cancer is a renal cell carcinoma (RCC). Inone embodiment, the renal cell carcinoma is selected from the groupconsisting of clear cell renal cell carcinoma, papillary renal cellcarcinoma, chromophobe renal cell carcinoma, other rare types of renalcell carcinoma (e.g., Collecting duct RCC, multilocular cystic RCC,medullary carcinoma, mucinous tubular and spindle cell carcinoma,neuroblastoma-associated RCC, unclassified renal cell carcinoma), andmetastatic RCC. In one embodiment the renal cancer is selected from thegroup consisting of transitional cell carcinoma, Wilms tumor(nephroblastoma), renal sarcoma, and benign (non-cancerous) kidneytumors, renal adenoma, oncocytoma, and angiomyolipoma.

The present disclosure also provides methods comprising combinationtherapy for the treatment of cancer. As used herein, “combinationtherapy” or “co-therapy” includes the administration of atherapeutically effective amount of apilimod as part of a specifictreatment regimen intended to provide the beneficial effect from theco-action of the apilimod and the additional active agent. The at leastone additional agent may be a therapeutic agent or a non-therapeuticagent. The beneficial effect of the combination includes, but is notlimited to, pharmacokinetic or pharmacodynamic co-action resulting fromthe combination of therapeutic compounds. The beneficial effect of thecombination may also relate to the mitigation of a toxicity, sideeffect, or adverse event associated with another agent in thecombination. “Combination therapy” is not intended to encompass theadministration of two or more of these therapeutic compounds as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin a beneficial effect that was not intended or predicted.

The at least one additional active agent may be a therapeutic agent, forexample an anti-cancer agent or a cancer chemotherapeutic agent, or anon-therapeutic agent, and combinations thereof. With respect totherapeutic agents, the beneficial effect of the combination includes,but is not limited to, pharmacokinetic or pharmacodynamic co-actionresulting from the combination of therapeutically active compounds. Withrespect to nontherapeutic agents, the beneficial effect of thecombination may relate to the mitigation of a toxicity, side effect, oradverse event associated with a therapeutically active agent in thecombination.

In one embodiment, the at least one additional agent is anon-therapeutic agent which mitigates one or more side effects of anapilimod composition, the one or more side effects selected from any ofnausea, vomiting, headache, dizziness, lightheadedness, drowsiness andstress. In one aspect of this embodiment, the non-therapeutic agent isan antagonist of a serotonin receptor, also known as 5-hydroxytryptaminereceptors or 5-HT receptors. In one aspect, the non-therapeutic agent isan antagonist of a 5-HT3 or 5-HT1a receptor. In one aspect, thenon-therapeutic agent is selected from the group consisting ofondansetron, granisetron, dolasetron and palonosetron. In anotheraspect, the non-therapeutic agent is selected from the group consistingof pindolol and risperidone.

In one embodiment, the at least one additional agent is a therapeuticagent. In one embodiment, the therapeutic agent is an anti-cancer agentas described in more detail below under ‘combination therapy’.

In the context of combination therapy, administration of apilimod, or apharmaceutically acceptable salt, solvate, clathrate, hydrate,polymorph, metabolite, prodrug, analog or derivative thereof, may besimultaneous with or sequential to the administration of the one or moreadditional active agents. In another embodiment, administration of thedifferent components of a combination therapy may be at differentfrequencies. The one or more additional agents may be administered priorto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a compound of the present disclosure.

The one or more additional active agents can be formulated forco-administration with apilimod in a single dosage form, as described ingreater detail herein. The one or more additional active agents can beadministered separately from the dosage form that comprises theapilimod. When the additional active agent is administered separatelyfrom the apilimod, it can be by the same or a different route ofadministration as the apilimod.

Preferably, the administration of a composition comprising apilimod incombination with one or more additional active agents provides asynergistic response in the subject being treated. In this context, theterm “synergistic” refers to the efficacy of the combination being moreeffective than the additive effects of either single therapy alone. Thesynergistic effect of a combination therapy according to the disclosurecan permit the use of lower dosages and/or less frequent administrationof at least one agent in the combination compared to its dose and/orfrequency outside of the combination. Additional beneficial effects ofthe combination can be manifested in the avoidance or reduction ofadverse or unwanted side effects associated with the use of eithertherapy in the combination alone (also referred to as monotherapy).

“Combination therapy” also embraces the administration of the compoundsof the present disclosure in further combination with non-drug therapies(e.g., surgery or radiation treatment). Where the combination therapyfurther comprises a non-drug treatment, the non-drug treatment may beconducted at any suitable time so long as a beneficial effect from theco-action of the combination of the therapeutic compounds and non-drugtreatment is achieved. For example, in appropriate cases, the beneficialeffect is still achieved when the non-drug treatment is temporallyremoved from the administration of the therapeutic compounds, perhaps bydays or even weeks.

The non-drug treatment can be selected from chemotherapy, radiationtherapy, hormonal therapy, anti-estrogen therapy, gene therapy, surgery(e.g. radical nephrectomy, partial nephrectomy, laparoscopic and roboticsurgery), radiofrequency ablation, and cryoablation. For example, anon-drug therapy is the removal of an ovary (e.g., to reduce the levelof estrogen in the body), thoracentesis (e.g., to remove fluid from thechest), paracentesis (e.g., to remove fluid from the abdomen), surgeryto remove or shrink angiomyolipomas, lung transplantation (andoptionally with an antibiotic to prevent infection due totransplantation), or oxygen therapy (e.g., through a nasal cannulacontaining two small plastic tubes or prongs that are placed in bothnostrils, through a face mask that fits over the nose and mouth, orthrough a small tube inserted into the windpipe through the front of theneck, also called transtracheal oxygen therapy).

In one embodiment, the at least one additional agent is an agent whichmitigates one or more side effects of apilimod selected from any ofnausea, vomiting, headache, dizziness, lightheadedness, drowsiness andstress. In one aspect of this embodiment, the additional agent is anantagonist of a serotonin receptors, also known as 5-hydroxytryptaminereceptors or 5-HT receptors. In one aspect, the additional agent is anantagonist of a 5-HT₃ or 5-HT_(1a) receptor. In one aspect, the agent isselected from the group consisting of ondansetron, granisetron,dolasetron and palonosetron. In another aspect, the agent is selectedfrom the group consisting of pindolol and risperidone.

In one embodiment, the at least one additional agent is an anti-canceragent. In one embodiment, the anti-cancer agent is selected from taxol,vincristine, doxorubicin, temsirolimus, carboplatin, ofatumumab,rituximab, and combinations thereof.

In one embodiment, the at least one additional agent is selected fromchlorambucil, ifosphamide, doxorubicin, mesalazine, thalidomide,lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib,paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone,CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin,or a combination thereof.

In one embodiment, the at least one additional agent is selected fromAfinitor (Everolimus), Aldesleukin, Avastin (Bevacizumab), Axitinib,Bevacizumab, Everolimus, IL-2 (Aldesleukin), Inlyta (Axitinib),Interleukin-2 (Aldesleukin), Nexavar (Sorafenib Tosylate), Pazopanib,Hydrochloride, Proleukin (Aldesleukin), Sorafenib Tosylate, SunitinibMalate, Sutent (Sunitinib Malate), Temsirolimus, Torisel (Temsirolimus),Votrient (Pazopanib Hydrochloride), or combination thereof.

In one embodiment, the at least one additional agent is directed towardstargeted therapy, wherein the treatment targets the cancer's specificgenes, proteins, or the tissue environment that contributes to cancergrowth and survival. This type of treatment blocks the growth and spreadof cancer cells while limiting damage to healthy cells.

In one embodiment, the at least one additional agent is directed towardsanti-angiogenesis therapy, wherein the treatment focuses on stoppingangiogenesis, which is the process of making new blood vessels. Becausea tumor needs the nutrients delivered by blood vessels to grow andspread, the goal of anti-angiogenesis therapies is to “starve” thetumor. One anti-angiogenic drug, bevacizumab (Avastin), has been shownto slow tumor growth for people with metastatic renal carcinoma.Bevacizumab combined with interferon slows tumor growth and spread.

In one embodiment, the at least one additional agent is directed towardsimmunotherapy, also called biologic therapy, is designed to boost thebody's natural defenses to fight cancer. It uses materials made eitherby the body or in a laboratory to improve, target, or restore immunesystem function. For example, Interleukin-2 (IL-2) is a drug that hasbeen used to treat kidney cancer as well as AM0010, and interleukin-15.They are cellular hormones called cytokines produced by white bloodcells and are important in immune system function, including thedestruction of tumor cells. Alpha-interferon is another type ofimmunotherapy used to treat kidney cancer that has spread. Interferonappears to change the proteins on the surface of cancer cells and slowtheir growth. Many combination therapies of IL-2 and alpha-interferonfor patients with advanced kidney cancer combined with chemotherapy aremore effective than IL-2 or interferon alone.

In embodiments, the at least one additional agent is a PD-1/PDL-1pathway inhibitor. In embodiments, the PD-1/PDL-1 pathway inhibitor isselected from pembrolizumab (Keytruda), avelumab, atezolizumab(MPDL3280A), nivolumab (BMS-936558), pidilizumab (MK-3475), MSB0010718C,and MEDI4736.

Another example is a compound called a check point inhibitor. Treatmentwith these compounds work by targeting molecules that serve as checksand balances on immune responses. By blocking these inhibitory moleculesor, alternatively, activating stimulatory molecules, these treatmentsare designed to unleash or enhance pre-existing anti-cancer immuneresponses. Various antibodies include PD-1, anti-CD27, B7-H3, KIR,LAG-3, 4-1BB/CD137, and GITR. Exemplary agents include pembrolizumab(Keytruda, MK-3475, a PD-1 antibody), MPDL3280A (a PD-L1 antibody),varlilumab (CDX-1127, an anti-CD27 antibody), MGA217 (an antibody thattargets B7-H3), lirilumab (a MR antibody), BMS-986016 (a LAG-3antibody), urelumab (a 4-1BB/CD137 antibody), TRX518 (a GITR antibody),and MK-4166 (a GITR antibody).

Another example is a cancer vaccine, designed to elicit an immuneresponse against tumor-specific or tumor-associated antigens,encouraging the immune system to attack cancer cells bearing theseantigens. Exemplary agents are AGS-003, DCVax, and NY-ESO-1.

Another example is immune cells are removed from a patient, geneticallymodified or treated with chemicals to enhance their activity, and thenre-introduced into the patient with the goal of improving the immunesystem's anti-cancer response.

In the context of the methods described herein, the amount of apilimodadministered to the subject is a therapeutically effective amount. Theterm “therapeutically effective amount” refers to an amount sufficientto treat, ameliorate a symptom of, reduce the severity of, or reduce theduration of the disease or disorder being treated or enhance or improvethe therapeutic effect of another therapy, or sufficient to exhibit adetectable therapeutic effect in the subject. In one embodiment, thetherapeutically effective amount of an apilimod composition is theamount effective to inhibit PIKfyve kinase activity.

An effective amount of apilimod can range from about 0.001 mg/kg toabout 1000 mg/kg, about 0.01 mg/kg to about 100 mg·kg, about 10 mg/kg toabout 250 mg/kg, about 0.1 mg/kg to about 15 mg/kg; or any range inwhich the low end of the range is any amount between 0.001 mg/kg and 900mg/kg and the upper end of the range is any amount between 0.1 mg/kg and1000 mg/kg (e.g., 0.005 mg/kg and 200 mg/kg, 0.5 mg/kg and 20 mg/kg).Effective doses will also vary, as recognized by those skilled in theart, depending on the diseases treated, route of administration,excipient usage, and the possibility of co-usage with other therapeutictreatments such as use of other agents. See, e.g., U.S. Pat. No.7,863,270, incorporated herein by reference.

In more specific aspects, the apilimod is administered at a dosageregimen of 30-1000 mg/day (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day)for at least 1 week (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 36,48, or more weeks). Preferably, apilimod is administered at a dosageregimen of 100-1000 mg/day for 4 or 16 weeks. Alternatively orsubsequently, apilimod is administered at a dosage regimen of 100-300 mgtwice a day for 8 weeks, or optionally, for 52 weeks. Alternatively orsubsequently, an apilimod composition is administered at a dosageregimen of 50 mg-1000 mg twice a day for 8 weeks, or optionally, for 52weeks.

An effective amount of the apilimod composition can be administered oncedaily, from two to five times daily, up to two times or up to threetimes daily, or up to eight times daily. In one embodiment, the apilimodcomposition is administered thrice daily, twice daily, once daily,fourteen days on (four times daily, thrice daily or twice daily, or oncedaily) and 7 days off in a 3-week cycle, up to five or seven days on(four times daily, thrice daily or twice daily, or once daily) and 14-16days off in 3 week cycle, or once every two days, or once a week, oronce every 2 weeks, or once every 3 weeks.

In accordance with the methods described herein, a “subject in needthereof” is a subject having renal cancer, or a subject having anincreased risk of developing renal cancer relative to the population atlarge. The subject in need thereof can be one that is “non-responsive”or “refractory” to a currently available therapy for the cancer. In thiscontext, the terms “non-responsive” and “refractory” refer to thesubject's response to therapy as not clinically adequate to relieve oneor more symptoms associated with the disease or disorder. In one aspectof the methods described here, the subject in need thereof is a subjecthaving cancer whose cancer is refractory to standard therapy or whosecancer has recurred following standard treatment.

A “subject” includes a mammal. The mammal can be e.g., any mammal, e.g.,a human, primate, vertebrate, bird, mouse, rat, fowl, dog, cat, cow,horse, goat, camel, sheep or a pig. Preferably, the mammal is a human.The term “patient” refers to a human subject.

The present disclosure also provides a monotherapy for the treatment ofrenal cancer as described herein. As used herein, “monotherapy” refersto the administration of a single active or therapeutic compound to asubject in need thereof.

As used herein, “treatment”, “treating” or “treat” describes themanagement and care of a patient for the purpose of combating a disease,condition, or disorder and includes the administration of apilimod toalleviate the symptoms or complications of a disease, condition ordisorder, or to eliminate the disease, condition or disorder.

As used herein, “prevention”, “preventing” or “prevent” describesreducing or eliminating the onset of the symptoms or complications ofthe disease, condition or disorder and includes the administration ofapilimod to reduce the onset, development or recurrence of symptoms ofthe disease, condition or disorder.

In one embodiment, the administration of apilimod leads to theelimination of a symptom or complication of the cancer being treated,however elimination of the cancer is not required. In one embodiment,the severity of the symptom is decreased. In the context of cancer, suchsymptoms may include clinical markers of severity or progressionincluding the degree to which a tumor secretes growth factors, degradesthe extracellular matrix, becomes vascularized, loses adhesion tojuxtaposed tissues, or metastasizes, as well as the number ofmetastases.

Treating cancer according to the methods described herein can result ina reduction in size of a tumor. A reduction in size of a tumor may alsobe referred to as “tumor regression”. Preferably, after treatment, tumorsize is reduced by 5% or greater relative to its size prior totreatment; more preferably, tumor size is reduced by 10% or greater;more preferably, reduced by 20% or greater; more preferably, reduced by30% or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75% or greater. Size of a tumor may be measured by anyreproducible means of measurement. The size of a tumor may be measuredas a diameter of the tumor.

Treating cancer according to the methods described herein can result ina reduction in tumor volume. Preferably, after treatment, tumor volumeis reduced by 5% or greater relative to its size prior to treatment;more preferably, tumor volume is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75% or greater. Tumor volume may be measured by anyreproducible means of measurement.

Treating cancer according to the methods described herein can result ina decrease in number of tumors. Preferably, after treatment, tumornumber is reduced by 5% or greater relative to number prior totreatment; more preferably, tumor number is reduced by 10% or greater;more preferably, reduced by 20% or greater; more preferably, reduced by30% or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. Number of tumors may be measured by any reproduciblemeans of measurement. The number of tumors may be measured by countingtumors visible to the naked eye or at a specified magnification.Preferably, the specified magnification is 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer according to the methods described herein can result ina decrease in number of metastatic lesions in other tissues or organsdistant from the primary tumor site. Preferably, after treatment, thenumber of metastatic lesions is reduced by 5% or greater relative tonumber prior to treatment; more preferably, the number of metastaticlesions is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75%. The number ofmetastatic lesions may be measured by any reproducible means ofmeasurement. The number of metastatic lesions may be measured bycounting metastatic lesions visible to the naked eye or at a specifiedmagnification. Preferably, the specified magnification is 2×, 3×, 4×,5×, 10×, or 50×.

Treating cancer according to the methods described herein can result inan increase in average survival time of a population of treated subjectsin comparison to a population receiving carrier alone. Preferably, theaverage survival time is increased by more than 30 days; morepreferably, by more than 60 days; more preferably, by more than 90 days;and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.An increase in average survival time of a population may be measured,for example, by calculating for a population the average length ofsurvival following initiation of treatment with an active compound. Anincrease in average survival time of a population may also be measured,for example, by calculating for a population the average length ofsurvival following completion of a first round of treatment with anactive compound.

Treating cancer according to the methods described herein can result inan increase in average survival time of a population of treated subjectsin comparison to a population of untreated subjects. Preferably, theaverage survival time is increased by more than 30 days; morepreferably, by more than 60 days; more preferably, by more than 90 days;and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.An increase in average survival time of a population may be measured,for example, by calculating for a population the average length ofsurvival following initiation of treatment with an active compound. Anincrease in average survival time of a population may also be measured,for example, by calculating for a population the average length ofsurvival following completion of a first round of treatment with anactive compound.

Treating cancer according to the methods described herein can result inincrease in average survival time of a population of treated subjects incomparison to a population receiving monotherapy with a drug that is notapilimod. Preferably, the average survival time is increased by morethan 30 days; more preferably, by more than 60 days; more preferably, bymore than 90 days; and most preferably, by more than 120 days. Anincrease in average survival time of a population may be measured by anyreproducible means. An increase in average survival time of a populationmay be measured, for example, by calculating for a population theaverage length of survival following initiation of treatment with anactive compound. An increase in average survival time of a populationmay also be measured, for example, by calculating for a population theaverage length of survival following completion of a first round oftreatment with an active compound.

Treating cancer according to the methods described herein can result ina decrease in the mortality rate of a population of treated subjects incomparison to a population receiving carrier alone. Treating a disorder,disease or condition according to the methods described herein canresult in a decrease in the mortality rate of a population of treatedsubjects in comparison to an untreated population. Treating a disorder,disease or condition according to the methods described herein canresult in a decrease in the mortality rate of a population of treatedsubjects in comparison to a population receiving monotherapy with a drugthat is not apilimod. Preferably, the mortality rate is decreased bymore than 2%; more preferably, by more than 5%; more preferably, by morethan 10%; and most preferably, by more than 25%. A decrease in themortality rate of a population of treated subjects may be measured byany reproducible means. A decrease in the mortality rate of a populationmay be measured, for example, by calculating for a population theaverage number of disease-related deaths per unit time followinginitiation of treatment with an active compound. A decrease in themortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active compound.

Treating cancer according to the methods described herein can result ina decrease in tumor growth rate. Preferably, after treatment, tumorgrowth rate is reduced by at least 5% relative to number prior totreatment; more preferably, tumor growth rate is reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. Tumor growthrate may be measured by any reproducible means of measurement. Tumorgrowth rate can be measured according to a change in tumor diameter perunit time. In one embodiment, after treatment the tumor growth rate maybe about zero and is determined to maintain the same size, e.g., thetumor has stopped growing.

Treating cancer according to the methods described herein can result ina decrease in tumor regrowth. Preferably, after treatment, tumorregrowth is less than 5%; more preferably, tumor regrowth is less than10%; more preferably, less than 20%; more preferably, less than 30%;more preferably, less than 40%; more preferably, less than 50%; evenmore preferably, less than 50%; and most preferably, less than 75%.Tumor regrowth may be measured by any reproducible means of measurement.Tumor regrowth is measured, for example, by measuring an increase in thediameter of a tumor after a prior tumor shrinkage that followedtreatment. A decrease in tumor regrowth is indicated by failure oftumors to reoccur after treatment has stopped.

Treating or preventing a cell proliferative disorder according to themethods described herein can result in a reduction in the rate ofcellular proliferation. Preferably, after treatment, the rate ofcellular proliferation is reduced by at least 5%; more preferably, by atleast 10%; more preferably, by at least 20%; more preferably, by atleast 30%; more preferably, by at least 40%; more preferably, by atleast 50%; even more preferably, by at least 50%; and most preferably,by at least 75%. The rate of cellular proliferation may be measured byany reproducible means of measurement. The rate of cellularproliferation is measured, for example, by measuring the number ofdividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder according to themethods described herein can result in a reduction in the proportion ofproliferating cells. Preferably, after treatment, the proportion ofproliferating cells is reduced by at least 5%; more preferably, by atleast 10%; more preferably, by at least 20%; more preferably, by atleast 30%; more preferably, by at least 40%; more preferably, by atleast 50%; even more preferably, by at least 50%; and most preferably,by at least 75%. The proportion of proliferating cells may be measuredby any reproducible means of measurement. Preferably, the proportion ofproliferating cells is measured, for example, by quantifying the numberof dividing cells relative to the number of nondividing cells in atissue sample. The proportion of proliferating cells can be equivalentto the mitotic index.

Treating or preventing a cell proliferative disorder according to themethods described herein can result in a decrease in the size of an areaor zone of cellular proliferation. Preferably, after treatment, size ofan area or zone of cellular proliferation is reduced by at least 5%relative to its size prior to treatment; more preferably, reduced by atleast 10%; more preferably, reduced by at least 20%; more preferably,reduced by at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. The size of anarea or zone of cellular proliferation may be measured by anyreproducible means of measurement. The size of an area or zone ofcellular proliferation may be measured as a diameter or width of an areaor zone of cellular proliferation.

Treating or preventing a cell proliferative disorder according to themethods described herein can result in a decrease in the number orproportion of cells having an abnormal appearance or morphology.Preferably, after treatment, the number of cells having an abnormalmorphology is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. An abnormal cellular appearance ormorphology may be measured by any reproducible means of measurement. Anabnormal cellular morphology can be measured by microscopy, e.g., usingan inverted tissue culture microscope. An abnormal cellular morphologycan take the form of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, apilimod actsselectively on a hyper-proliferating cells or abnormally proliferatingcells, compared to normal cells. As used herein, a “normal cell” is acell that cannot be classified as part of a “cell proliferativedisorder”. A normal cell lacks unregulated or abnormal growth, or both,that can lead to the development of an unwanted condition or disease.Preferably, a normal cell possesses normally functioning cell cyclecheckpoint control mechanisms. Preferably, apilimod, acts selectively tomodulate one molecular target (e.g., a target kinase) but does notsignificantly modulate another molecular target (e.g., a non-targetkinase). The disclosure also provides a method for selectivelyinhibiting the activity of an enzyme, such as a kinase. Preferably, anevent occurs selectively in population A relative to population B if itoccurs greater than two times more frequently in population A ascompared to population B. An event occurs selectively if it occursgreater than five times more frequently in population A. An event occursselectively if it occurs greater than ten times more frequently inpopulation A; more preferably, greater than fifty times; even morepreferably, greater than 100 times; and most preferably, greater than1000 times more frequently in population A as compared to population B.For example, cell death would be said to occur selectively in diseasedor hyper-proliferating cells if it occurred greater than twice asfrequently in diseased or hyper-proliferating cells as compared tonormal cells.

Pharmaceutical Compositions and Formulations

The present disclosure provides pharmaceutical compositions comprisingan amount of apilimod, or a pharmaceutically acceptable salt, solvate,clathrate, hydrate, polymorph, metabolite, prodrug, analog or derivativethereof, in combination with at least one pharmaceutically acceptableexcipient or carrier, wherein the amount is effective for the treatmentof renal cancer and/or effective to inhibit PIKfyve in the cancer cellsof a subject having cancer.

In one embodiment, the apilimod is apilimod free base. In oneembodiment, the apilimod is apilimod dimesylate.

In one embodiment, the apilimod is combined with at least one additionalactive agent in a single dosage form. In one embodiment, the compositionfurther comprises an antioxidant.

In embodiments, the at least one additional active agent is selectedfrom the group consisting of an alkylating agent, an intercalatingagent, a tublin binding agent, a corticosteroid, and combinationsthereof. In one embodiment, the at least one additional active agent isa therapeutic agent selected from the group consisting of ibrutinib,rituximab, doxorubicin, prednisolone, vincristine, velcade, andeverolimus, and combinations thereof. In one embodiment, the at leastone additional active agent is a therapeutic agent selected fromcyclophosphamide, hydroxydaunorubicin (also referred to as doxorubicinor Adriamycin™), vincristine (also referred to as Oncovin™), prednisone,prednisolone, and combinations thereof.

In embodiments, the at least one additional active agent is anon-therapeutic agent selected to ameliorate one or more side effects ofthe apilimod composition. In one embodiment, the nontherapeutic agent isselected from the group consisting of ondansetron, granisetron,dolasetron and palonosetron. In one embodiment, the non-therapeuticagent is selected from the group consisting of pindolol and risperidone.

In embodiments, at least one additional agent is a PD-1/PDL-1 pathwayinhibitor. In embodiments, the PD-1/PDL-1 pathway inhibitor is selectedfrom pembrolizumab (Keytruda), avelumab, atezolizumab (MPDL3280A),nivolumab (BMS-936558), pidilizumab (MK-3475), MSB0010718C, andMEDI4736.

In embodiments, the at least one additional active agent is selectedfrom an inhibitor of the mTOR pathway, a TKI inhibitor, a PI3Kinhibitor, a dual PI3K/mTOR inhibitor, a SRC inhibitor, a VEGFinhibitor, a Janus kinase (JAK) inhibitor, a Raf inhibitor, an Erkinhibitor, a farnesyltransferase inhibitor, a histone deacetylaseinhibitor, an anti-mitotic agent, a multi-drug resistance effluxinhibitor, an antibiotic, and a cytokine. In one embodiment, the secondtherapeutic agent is a therapeutic cytokine. In one embodiment, thesecond therapeutic agent is Interleukin-2. In another embodiment, thesecond therapeutic agent is selected from a tyrosine kinase inhibitor(e.g., everolimus, bevacizumab).

In embodiments, the mTOR inhibitor is selected from the group consistingof rapamycin (also referred to as sirolimus), everolimus, temsirolimus,ridaforolimus, umirolimus, zotarolimus, AZD8055, INK128, WYE-132,Torin-1, pyrazolopyrimidine analogs PP242, PP30, PP487, PP121,KU0063794, KU-BMCL-200908069-1, Wyeth-BMCL-200910075-9b, INK-128, XL388,AZD8055, P2281, and P529. See, e.g., Liu et al. Drug Disc. Today Ther.Strateg., 6(2): 47-55 (2009).

In embodiments, the mTOR inhibitor istrans-4-[4-amino-5-(7-methoxy-1H-indol-2-yl)imidazo[5,1-f][1,2,4]triazin-7-yl]cyclohexanecarboxylic acid (also known as OSI-027), and any salts, solvates,hydrates, and other physical forms, crystalline or amorphous, thereof.See US 2007/0112005. OSI-027 can be prepared according to US2007/0112005, incorporated herein by reference. In one embodiment, themTOR inhibitor is OXA-01. See e.g., WO 2013152342 A1.

In embodiments, the PI3K inhibitor is selected from the group consistingof GS-1101 (Idelalisib), GDC0941 (Pictilisib), LY294002, BKM120(Buparlisib), PI-103, TGX-221, IC-87114, XL 147, ZSTK474, BYL719,AS-605240, PIK-75, 3-methyladenine, A66, PIK-93, PIK-90, AZD6482,IPI-145 (Duvelisib), TG100-115, AS-252424, PIK294, AS-604850,GSK2636771, BAY 80-6946 (Copanlisib), CH5132799, CAY10505, PIK-293,TG100713, CZC24832 and HS-173.

In embodiments, the dual PI3K/mTOR inhibitor is selected from the groupconsisting of, GDC-094, WAY-001, WYE-354, WAY-600, WYE-687,Wyeth-BMCL-200910075-16b, Wyeth-BMCL-200910096-27, KU0063794 andKUBMCL-200908069-5, NVP-BEZ235, XL-765, PF-04691502, GDC-0980(Apitolisib), GSK1059615, PF-05212384, BGT226, PM-402, VS-558 andGSK2126458. See, e.g., Liu et al. Drug Disc. Today Ther. Strateg., 6(2):47-55 (2009), incorporated herein by reference.

In embodiments, the mTOR pathway inhibitor is a polypeptide (e.g., anantibody or fragment thereof) or a nucleic acid (e.g., a double-strandedsmall interfering RNA, a short hairpin RNA, a micro-RNA, an antisenseoligonucleotide, a locked nucleic acid, or an aptamer) that binds to andinhibits the expression level or activity or a protein (or nucleic acidencoding the protein) in the mTOR pathway. For example, the polypeptideor nucleic acid inhibits mTOR Complex 1 (mTORC1), regulatory-associatedprotein of mTOR (Raptor), mammalian lethal with SEC13 protein 8 (MLST8),proline-rich Akt substrate of 40 kDa (PRAS40), DEP domain-containingmTOR-interacting protein (DEPTOR), mTOR Complex 2 (mTORC2),rapamycin-insensitive companion of mTOR (RICTOR), G protein betasubunit-like (GβL), mammalian stress-activated protein kinaseinteracting protein 1 (mSIN1), paxillin, RhoA, Ras-related C3 botulinumtoxin substrate 1 (Rad), Cell division control protein 42 homolog(Cdc42), protein kinase C α (PKCα), the serine/threonine protein kinaseAkt, phosphoinositide 3-kinase (PI3K), p70S6K, Ras, and/or eukaryotictranslation initiation factor 4E (eIF4E)-binding proteins (4EBPs), orthe nucleic acid encoding one of these proteins.

In embodiments, the SRC inhibitor is selected from the group consistingof bosutinib, saracatinib, dasatinib, ponatinib, KX2-391, XL-228,TG100435/TG100855, and DCC2036. See, e.g., Puls et al. Oncologist. 2011May; 16(5): 566-578. In one embodiment, the SRC inhibitor is apolypeptide (e.g., an antibody or fragment thereof) or nucleic acid(e.g., a double-stranded small interfering RNA, a short hairpin RNA, amicro-RNA, an antisense oligonucleotide, a locked nucleic acid, or anaptamer) that binds to and inhibits the expression level or activity ofthe SRC protein or a nucleic acid encoding the SRC protein.

In embodiments, the VEGF inhibitor is selected from bevacizumab,sunitinib, pazopanib, axitinib, sorafenib, regorafenib, lenvatinib, andmotesanib. In one embodiment, the VEGF inhibitor is a polypeptide (e.g.,an antibody or fragment thereof) or nucleic acid (e.g., adouble-stranded small interfering RNA, a short hairpin RNA, a micro-RNA,an antisense oligonucleotide, a morpholino, a locked nucleic acid, or anaptamer) that binds to and inhibits the expression level or activity ofa VEGF protein, a VEGF receptor protein, or a nucleic acid encoding oneof these proteins. For example, the VEGF inhibitor is a soluble VEGFreceptor (e.g., a soluble VEGF-C/D receptor (sVEGFR-3)).

In embodiments, the JAK inhibitor is selected from facitinib,ruxolitinib, baricitinib, CYT387 (CAS number 1056634-68-4),lestaurtinib, pacritinib, and TG101348 (CAS number 936091-26-8). In oneembodiment, the JAK inhibitor is a polypeptide (e.g., an antibody orfragment thereof) or nucleic acid (e.g., a double-stranded smallinterfering RNA, a short hairpin RNA, a micro-RNA, an antisenseoligonucleotide, a morpholino, a locked nucleic acid, or an aptamer)that binds to and inhibits the expression level or activity of a JAK(e.g., JAK1, JAK2, JAK3, or TYK2) or a nucleic acid encoding the JAKprotein.

In embodiments, the Raf inhibitor is selected from PLX4032(vemurafenib), sorafenib, PLX-4720, GSK2118436 (dabrafenib), GDC-0879,RAF265, AZ 628, NVP-BHG712, SB90885, ZM 336372, GW5074, TAK-632,CEP-32496 and LGX818 (Encorafenib). In one embodiment, the Raf inhibitoris a polypeptide (e.g., an antibody or fragment thereof) or nucleic acid(e.g., a double-stranded small interfering RNA, a short hairpin RNA, amicro-RNA, an antisense oligonucleotide, a morpholino, a locked nucleicacid, or an aptamer) that binds to and inhibits the expression level oractivity of a Raf (e.g., A-Raf, B-Raf, C-Raf) or a nucleic acid encodingthe Raf protein. In one embodiment, the MEK inhibitor is selected fromAZD6244 (Selumetinib), PD0325901, GSK1120212 (Trametinib), U0126-EtOH,PD184352, RDEA119 (Rafametinib), PD98059, BIX 02189, MEK162(Binimetinib), AS-703026 (Pimasertib), SL-327, BIX02188, AZD8330,TAK-733 and PD318088. In one embodiment, the MEK inhibitor is apolypeptide (e.g., an antibody or fragment thereof) or nucleic acid(e.g., a double-stranded small interfering RNA, a short hairpin RNA, amicro-RNA, an antisense oligonucleotide, a morpholino, a locked nucleicacid, or an aptamer) that binds to and inhibits the expression level oractivity of a MEK (e.g., MEK-1, MEK-2) or a nucleic acid encoding theMEK protein.

In embodiments, the Akt inhibitor is selected from MK-2206, KRX-0401(perifosine), GSK690693, GDC-0068 (Ipatasertib), AZD5363, CCT128930,A-674563, PHT-427. In one embodiment, the Akt inhibitor is a polypeptide(e.g., an antibody or fragment thereof) or nucleic acid (e.g., adouble-stranded small interfering RNA, a short hairpin RNA, a micro-RNA,an antisense oligonucleotide, a morpholino, a locked nucleic acid, or anaptamer) that binds to and inhibits the expression level or activity ofa Akt (e.g., Akt-1, Akt-2, Akt-3) or a nucleic acid encoding the Aktprotein.

In embodiments, the famesyltransferase inhibitor is selected fromLB42708 or tipifarnib. In one embodiment, the farnesyltransferaseinhibitor is a polypeptide (e.g., an antibody or fragment thereof) ornucleic acid (e.g., a double-stranded small interfering RNA, a shorthairpin RNA, a micro-RNA, an antisense oligonucleotide, a morpholino, alocked nucleic acid, or an aptamer) that binds to and inhibits theexpression level or activity of farnesyltransferase or a nucleic acidencoding the farnesyltransferase protein. In one embodiment, the histonemodulating inhibitor is selected from anacardic acid, C646, MG149(histone acetyltransferase), GSK J4 Hcl (histone demethylase), GSK343(active against EZH2), BIX 01294 (histone methyltransferase), MK0683(Vorinostat), MS275 (Entinostat), LBH589 (Panobinostat), Trichostatin A,MGCD0103 (Mocetinostat), Tasquinimod, TMP269, Nexturastat A, RG2833,PDX101 (Belinostat).

In embodiments, the anti-mitotic agent is selected from Griseofulvin,vinorelbine tartrate, paclitaxel, docetaxel, vincristine, vinblastine,Epothilone A, Epothilone B, ABT-751, CYT997 (Lexibulin), vinfluninetartrate, Fosbretabulin, GSK461364, ON-01910 (Rigosertib), Ro3280,BI2536, NMS-P937, BI 6727 (Volasertib), HMN-214 and MLN0905.

In embodiments, the tyrosine kinase inhibitor is selected from Votrient,Axitinib, Bortezomib, Bosutinib, Carfilzomib, Crizotinib, Dabrafenib,Dasatinib, Erlotinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib,Nilotinib, Pegaptanib, Ponatinib, Regorafenib, Ruxolitinib, Sorafenib,Sunitinib, Trametinib, Vandetanib, Vemurafenib, and Vismodegib.

In one embodiment, the polyether antibiotic is selected from sodiummonensin, nigericin, valinomycin, salinomycin.

A “pharmaceutical composition” is a formulation containing the compoundsdescribed herein in a pharmaceutically acceptable form suitable foradministration to a subject. As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, materials,compositions, carriers, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. Examples of pharmaceutically acceptableexcipients include, without limitation, sterile liquids, water, bufferedsaline, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like), oils, detergents, suspending agents,carbohydrates (e.g., glucose, lactose, sucrose or dextran), antioxidants(e.g., ascorbic acid or glutathione), chelating agents, low molecularweight proteins, or suitable mixtures thereof.

A pharmaceutical composition can be provided in bulk or in dosage unitform. It is especially advantageous to formulate pharmaceuticalcompositions in dosage unit form for ease of administration anduniformity of dosage. The term “dosage unit form” as used herein refersto physically discrete units suited as unitary dosages for the subjectto be treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved. A dosage unit form can bean ampoule, a vial, a suppository, a dragee, a tablet, a capsule, an IVbag, or a single pump on an aerosol inhaler.

In therapeutic applications, the dosages vary depending on the agent,the age, weight, and clinical condition of the recipient patient, andthe experience and judgment of the clinician or practitioneradministering the therapy, among other factors affecting the selecteddosage. Generally, the dose should be a therapeutically effectiveamount. Dosages can be provided in mg/kg/day units of measurement (whichdose may be adjusted for the patient's weight in kg, body surface areain m², and age in years). An effective amount of a pharmaceuticalcomposition is that which provides an objectively identifiableimprovement as noted by the clinician or other qualified observer. Forexample, alleviating a symptom of a disorder, disease or condition. Asused herein, the term “dosage effective manner” refers to amount of apharmaceutical composition to produce the desired biological effect in asubject or cell.

For example, the dosage unit form can comprise 1 nanogram to 2milligrams, or 0.1 milligrams to 2 grams; or from 10 milligrams to 1gram, or from 50 milligrams to 500 milligrams or from 1 microgram to 20milligrams; or from 1 microgram to 10 milligrams; or from 0.1 milligramsto 2 milligrams.

The pharmaceutical compositions can take any suitable form (e.g,liquids, aerosols, solutions, inhalants, mists, sprays; or solids,powders, ointments, pastes, creams, lotions, gels, patches and the like)for administration by any desired route (e.g, pulmonary, inhalation,intranasal, oral, buccal, sublingual, parenteral, subcutaneous,intravenous, intramuscular, intraperitoneal, intrapleural, intrathecal,transdermal, transmucosal, rectal, and the like). For example, apharmaceutical composition of the disclosure may be in the form of anaqueous solution or powder for aerosol administration by inhalation orinsufflation (either through the mouth or the nose), in the form of atablet or capsule for oral administration; in the form of a sterileaqueous solution or dispersion suitable for administration by eitherdirect injection or by addition to sterile infusion fluids forintravenous infusion; or in the form of a lotion, cream, foam, patch,suspension, solution, or suppository for transdermal or transmucosaladministration.

A pharmaceutical composition can be in the form of an orally acceptabledosage form including, but not limited to, capsules, tablets, buccalforms, troches, lozenges, and oral liquids in the form of emulsions,aqueous suspensions, dispersions or solutions. Capsules may containmixtures of a compound of the present disclosure with inert fillersand/or diluents such as the pharmaceutically acceptable starches (e.g.,corn, potato or tapioca starch), sugars, artificial sweetening agents,powdered celluloses, such as crystalline and microcrystallinecelluloses, flours, gelatins, gums, etc. In the case of tablets for oraluse, carriers which are commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, can also be added. Fororal administration in a capsule form, useful diluents include lactoseand dried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the compound of the present disclosure may besuspended or dissolved in an oily phase is combined with emulsifyingand/or suspending agents. If desired, certain sweetening and/orflavoring and/or coloring agents may be added.

A pharmaceutical composition can be in the form of a tablet. The tabletcan comprise a unit dosage of a compound of the present disclosuretogether with an inert diluent or carrier such as a sugar or sugaralcohol, for example lactose, sucrose, sorbitol or mannitol. The tabletcan further comprise a non-sugar derived diluent such as sodiumcarbonate, calcium phosphate, calcium carbonate, or a cellulose orderivative thereof such as methyl cellulose, ethyl cellulose,hydroxypropyl methyl cellulose, and starches such as corn starch. Thetablet can further comprise binding and granulating agents such aspolyvinylpyrrolidone, disintegrants (e.g. swellable crosslinked polymerssuch as crosslinked carboxymethylcellulose), lubricating agents (e.g.stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT),buffering agents (for example phosphate or citrate buffers), andeffervescent agents such as citrate/bicarbonate mixtures.

The tablet can be a coated tablet. The coating can be a protective filmcoating (e.g. a wax or varnish) or a coating designed to control therelease of the active agent, for example a delayed release (release ofthe active after a predetermined lag time following ingestion) orrelease at a particular location in the gastrointestinal tract. Thelatter can be achieved, for example, using enteric film coatings such asthose sold under the brand name Eudragit®.

Tablet formulations may be made by conventional compression, wetgranulation or dry granulation methods and utilize pharmaceuticallyacceptable diluents, binding agents, lubricants, disintegrants, surfacemodifying agents (including surfactants), suspending or stabilizingagents, including, but not limited to, magnesium stearate, stearic acid,talc, sodium lauryl sulfate, microcrystalline cellulose,carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginicacid, acacia gum, xanthan gum, sodium citrate, complex silicates,calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalciumphosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride,talc, dry starches and powdered sugar. Preferred surface modifyingagents include nonionic and anionic surface modifying agents.Representative examples of surface modifying agents include, but are notlimited to, poloxamer 188, benzalkonium chloride, calcium stearate,cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters,colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesiumaluminum silicate, and triethanolamine.

A pharmaceutical composition can be in the form of a hard or softgelatin capsule. In accordance with this formulation, the compound ofthe present disclosure may be in a solid, semi-solid, or liquid form.

A pharmaceutical composition can be in the form of a sterile aqueoussolution or dispersion suitable for parenteral administration. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intra-articular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques.

A pharmaceutical composition can be in the form of a sterile aqueoussolution or dispersion suitable for administration by either directinjection or by addition to sterile infusion fluids for intravenousinfusion, and comprises a solvent or dispersion medium containing,water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, or one or morevegetable oils. Solutions or suspensions of the compound of the presentdisclosure as a free base or pharmacologically acceptable salt can beprepared in water suitably mixed with a surfactant. Examples of suitablesurfactants are given below. Dispersions can also be prepared, forexample, in glycerol, liquid polyethylene glycols and mixtures of thesame in oils.

The pharmaceutical compositions for use in the methods of the presentdisclosure can further comprise one or more additives in addition to anycarrier or diluent (such as lactose or mannitol) that is present in theformulation. The one or more additives can comprise or consist of one ormore surfactants. Surfactants typically have one or more long aliphaticchains such as fatty acids which enables them to insert directly intothe lipid structures of cells to enhance drug penetration andabsorption. An empirical parameter commonly used to characterize therelative hydrophilicity and hydrophobicity of surfactants is thehydrophilic-lipophilic balance (“HLB” value). Surfactants with lower HLBvalues are more hydrophobic, and have greater solubility in oils, whilesurfactants with higher HLB values are more hydrophilic, and havegreater solubility in aqueous solutions. Thus, hydrophilic surfactantsare generally considered to be those compounds having an HLB valuegreater than about 10, and hydrophobic surfactants are generally thosehaving an HLB value less than about 10. However, these HLB values aremerely a guide since for many surfactants, the HLB values can differ byas much as about 8 HLB units, depending upon the empirical method chosento determine the HLB value.

Among the surfactants for use in the compositions of the disclosure arepolyethylene glycol (PEG)-fatty acids and PEG-fatty acid mono anddiesters, PEG glycerol esters, alcohol-oil transesterification products,polyglyceryl fatty acids, propylene glycol fatty acid esters, sterol andsterol derivatives, polyethylene glycol sorbitan fatty acid esters,polyethylene glycol alkyl ethers, sugar and its derivatives,polyethylene glycol alkyl phenols, polyoxyethylene-polyoxypropylene(POE-POP) block copolymers, sorbitan fatty acid esters, ionicsurfactants, fat-soluble vitamins and their salts, water-solublevitamins and their amphiphilic derivatives, amino acids and their salts,and organic acids and their esters and anhydrides.

The present disclosure also provides packaging and kits comprisingpharmaceutical compositions for use in the methods of the presentdisclosure. The kit can comprise one or more containers selected fromthe group consisting of a bottle, a vial, an ampoule, a blister pack,and a syringe. The kit can further include one or more of instructionsfor use in treating and/or preventing a disease, condition or disorderof the present disclosure, one or more syringes, one or moreapplicators, or a sterile solution suitable for reconstituting apharmaceutical composition of the present disclosure.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present disclosure areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentdisclosure. The examples do not limit the claimed disclosure. Based onthe present disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present disclosure.

EXAMPLES Example 1: Apilimod is a Highly Selective Inhibitor of TSC2Null Cell Proliferation

Apilimod was identified in a high throughput cell viability screen usingTSC2−/− mouse embryonic fibroblasts (MEF-EV) cells. TSC2 null cells haveconstitutively active mTOR. Briefly, MEF cells derived from TSC2−/−knockout mouse embryos (Onda et al., J. Clin. Invest. 104(6):687-95,1999) were infected with a retrovirus vector encoding the hygromycinantibiotic resistance gene (MEF-EV) or the same retrovirus vector alsoencoding TSC2 (MEF-TSC2). The MEF-EV and MEF-TSC2 line were thenestablished by hygromycin selection.

Cells were expanded in DMEM containing 10% FBS (Omega Scientific) and 2mM L-Glutamine. Frozen stocks of cells were prepared for direct use inthe HTS assay. Cells were harvested, pelleted and then resuspended in95% FBS & 5% DMSO at a concentration 1×10⁷ cells/ml., One ml aliquotswere rate frozen to −80 at a rate of 1 degree per minute. These stockswere then transferred to vapor phase liquid nitrogen for long termstorage.

For screening, vials were thawed at 37° C. with continuous agitationuntil just thawed then re-suspended in room temperature assay media andcentrifuged at 1,000 rpm for 5 minutes. The resulting pellet wasre-suspended in appropriate volume and counted using an automated cellcounter and diluted accordingly to a final count of 40,000 cells/ml.

Test compounds (5 μl stock solution, 6× desired final wellconcentration) were dispensed to 384-well assay plates (Corning 3712)using a Biomek FX liquid handler. MEF-EV cells (1000 cells per well in25 μL of media) were added to these pre-formatted plates using a ThermoWellmate, non-contact dispensing system with a standard bore cassettehead. Plates were incubated for 72h at 37° C. under an atmosphere of 5%CO₂ in a humidified incubator.

Cell viability was determined with CellTiter-Glo® luminescence assay(Promega) as per the manufacturer's instructions. Viability wasexpressed as a percentage of untreated control cells. As an example, forapilimod, MEF-EV cell viability (Mean+/−StDev, n=3) was 2.16+/−0.36% @0.5 μM and 1.94+/−0.07% @ 5 μM.

The activity of apilimod on TSC2 deficient cells was furtherdemonstrated by performing 10 point dose response on the MEF-EV andMEF-TSC2 lines described above as well as three additional pairs ofisogenic lines: (1) (TSC2−/−, p53−/−) and (TSC2+/−, p53−/−) MEF lineswere established from (TSC2−/−, p53−/−) or (TSC2+/−, p53−/−) embryosaccording to standard methods. See e.g., Zhang et al. J. Clin. Invest.112, 1223-33, 2003. (2) ELT3-EV and ELT3-TSC2 lines were establishedfrom the ELT3 rat tumor cell line. The ELT3 line is an established rattumor model for LAM/TSC. See e.g., Howe et al., Am. J. Path. 146,1568-79, 1995. These cells harbor an inactivating mutation in TSC2,which leads to constitutive activation of the mTOR pathway. To developan isogenic pair of cells ELT3 cells were infected with a retrovirusvector encoding the hygromycin antibiotic resistance gene (ELT3-EV) orthe same retrovirus vector also encoding TSC2 (ELT3-TSC2). The ELT3-EVand ELT3-TSC2 line were then established by hygromycin selection. (3)TRI-AML102 and AML103 lines were established from a TSC2 null primaryhuman AML sample provided by Dr. Elizabeth Henske (Fox Chase CancerCenter, Philadelphia, Pa.). The cells were infected with amphotropicretrovirus LXSN16E6E7 that encodes the HPV16 E6 and E7 open readingframes and neomycin resistance cassette. Cells were expanded andneomycin-selected. Individual clones were isolated and frozen down. Thecoding sequence for the human Telomerase gene (hTERT) with hygromycinresistance cassette (pLXSN hTERT-hyg plasmid) was stably expressed intoa TSC2^(−/−) confirmed E6E7 AML clone using Fugene6 transfection reagent(Roche Applied Science, Indianapolis, Ind.). TRI-AML102 was generated bystable incorporation of a control zeomycin selection plasmid(pcDNA3.1-zeo), while TRI-AML103 expresses the human TSC2 cDNApcDNA3.1-zeo plasmid. As a result of these engineering processes, bothTRI102 and TRI103 are neomycin, hygromycin, and zeomycin resistantlines.

For 10-point dose response, 750 MEF, 2000 ELT3, or 2000 AML cells in 100μL of growth media (DMEM (CellGro 10-017-CV) FBS 10% (Sigma AldrichF2442-500ML, Lot 12D370) Penicillin/Streptomycin (100×) (CellGro Ref30-002) were plated per well of a 96 well plate. 24 hours after platingcells, the media was removed and apilimod dilutions (1-500 nM, 2-folddilutions) in 100 μL of growth media were added (0.1% final DMSOconcentration). 72 hours after compound addition, relative cellviability was determined by CellTiter-Glo® luminescence assay (Promega)and expressed as a percentage relative to vehicle (DMSO) treated controlcells. IC₅₀ values were then calculated from the dose response curvesusing XLFIT (IDBS).

The TSC2 deficient cells were highly sensitive to apilimod (IC₅₀=20 nM,FIG. 1). TSC2−/− p53−/− MEFs demonstrated increased sensitivity toapilimod compared to the TSC2+/−p53−/− MEFs as indicated by aselectivity ratio above 1 (2.45).

TABLE 1 IC₅₀ (viability) of apilimod in various cell types Cell type:MEF AML MEF TSC2−/− TSC2+/− TSC2−/− p53−/− p53−/− ELT3 IC50 TSC2−/−19.70 28.80 117.00 13.70 IC50 TSC2 rescue 20.10 70.70 132.00 16.05Selectivity Ratio 1.02 2.45 1.13 1.17 IC50s (nM) calculated from10-point dose response on TSC2 −/− deficient and rescue lines. IC50s arecalculated from the average of two experiments. The selectivity ratio iscalculated by dividing the IC50 of the TSC2 rescue line by the TSC2−/−line.

Furthermore, higher concentrations of apilimod had higher potency on theTSC2−/− MEF-EV cells compared to the TSC2 rescue MEF-TSC2 cells. Thisdata, coupled to the fact that apilimod is not cytotoxic on peripheralblood mononuclear cells (Wada et al., Blood 109, 1156-64, 2007), nor ona variety of other cancer lines including U937, HELA, Jurkat, and THP-1(PCT Publication No. WO 2006/128129) suggests that there will be a hightherapeutic index for treating TSC2−/− cancer cells with apilimod (FIG.2A-2B).

Example 2: Apilimod is a Highly Selective Cytotoxic Agent in CancerCells

The cytotoxic activity of apilimod was evaluated using a standard cellviability assay such as CellTiterGlo™ according to the manufacturer'sinstructions. 122 human cancer cell lines were evaluated for sensitivityto apilimod. A cell line was called as apilimod sensitive if the IC₅₀was less than 500 nM. 35 cell lines were identified as sensitive toapilimod-induced cytotoxicity. Apilimod was also highly selective forcancer cells compared to normal cells, which had IC₅₀'s ranging from20-200 fold higher than the cancer cells (FIGS. 2A-2B).

The mechanism of apilimod's cytotoxic activity was further investigatedby assaying for autophagic vacuoles after 72 hours of treatment in an H4neuroglioma cell line (IC₅₀ 250-300 nM). Autophagy was quantified usingthe Cyto-ID Autophagy detection kit (Enzo) according to manufacturer'sdirections. FIG. 3 shows that apilimod induced autophagy in adose-dependent manner.

Example 3: Apilimod is a Highly Selective Binder of PIKfyve Kinase

In order to identify the cellular target of apilimod in cancer cells,whole cell lysate prepared from human neuroglioma cells was used toidentify its binding partners using chemical capture mass spectrometry(CCMS). This work was performed at Caprotec Bioanalytics GmbH, BerlinGermany. See Michaelis et al., J. Med. Chem., 55 3934-44 (2012) andreferences cited therein.

Briefly, two capture compound variants employing apilimod as selectivityfunction attached in a single orientation were synthesized and analyzedby LC-MS and 1H-NMR to ensure identity and purity. Capture conditionswere optimized in whole cell lysate from H4 (human neuroglioma) cancercells, e.g. minimization of non-specific interactions of the proteinswith capture compounds, concentration of reagents and proteins to obtainmaximum binding of proteins and capture compounds, etc. One capturecompound was selected to identify specific protein binders in the CCMSexperiments using apilimod as a competitor ligand. Proteins that aredetected by LC-S in the capture assay and that are significantlydiminished in competition control experiments are considered to bespecific binders. These specific binders were further subjected tostringent data analysis criteria to determine specificity after unbiaseddata evaluation. Specific protein binders were ranked according to theirfold change (FC) values in the capture experiments. Only two proteinswere identified as high probability candidate target proteins ofapilimod: PIKfyve and Vac14. Volcano plot is shown in FIG. 4. FC andp-values for these proteins in the four different capture compoundconcentration experiments are shown in Table 2.

TABLE 2 Capture Compound Concentrations 0.1 μM 0.5 μM 1.0 μM 2.0 μMPIKfyve log₂ (FC) 6.3 6.2 4.1 4.3 −log₁₀ (p-value) 3.7 2.8 5.1 3.9 Vac14log (FC) 6.2 5.6 Inf. 3.9 −log₁₀ (p-value) 3.9 3.8 1.9 3.6

In a separate study, protein kinase profiling of apilimod was conductedto identify kinase targets (DiscoveRx, Fremont, Calif.). A dissociationconstant (K_(d)) study was performed using apilimod at increasingconcentrations (0.05-3000 nM) against PIKfyve, a known target ofapilimod. The experiment was performed in duplicate and the K_(d) wasdetermined to be 0.075 nM (range 0.069-0.081 nM) (FIG. 5).

Next, apilimod was screened against a comprehensive panel of kinases(PIKfyve not included). In total, 456 kinases, includingdisease-relevant kinases, were assayed for their ability to bind withapilimod. The screening concentration of apilimod was 1 μM, aconcentration that is >10,000 times greater than the K_(d) for apilimodagainst PIKfyve. The results from the screen showed that apilimod didnot bind to any of the 456 kinases tested.

Together, these results demonstrate that apilimod binds with highselectivity in cancer cells to a single cellular kinase, PIKfyve.PIKfyve is an enzyme that binds to PI(3)P and catalyzes the formation ofthe lipid second messengers PI(3,5)P2 and PI(5)P and others have shownthat apilimod is also a potent and specific inhibitor of this kinasePIKfyve in normal cells. Cai X et al., Chem Biol. 2013 Jul. 25;20(7):912-21. As discussed in more detail below, in order to understandthe mechanism of apilimod's selective cytotoxicity against cancer cells,we conducted a series of experiments aimed at elucidating its biologicalactivity in cancer cells.

Example 4: Mechanism of Anti-Cancer Activity of Apilimod

Apilimod was known to be a potent inhibitor of the inflammatorycytokines IL-12 and IL-23. To the extent apilimod was indicated fortreating a disease or disorder, it was predicated on this activity.Although the clinical testing of apilimod focused on its potentialefficacy in autoimmune and inflammatory diseases such as psoriasis,rheumatoid arthritis, and Crohn's disease, there were a few publishedsuggestions that apilimod might be useful against cancers, andspecifically against cancers in which c-rel or IL-12/23 were acting aspro-proliferative factors. See e.g., WO 2006/128129 and Baird et al.,Frontiers in Oncology 3:1 (2013), respectively. Surprisingly, andcontrary to these expectations predicated on apilimod's IL-12/23inhibitory activity, we found no correlation between any of c-Relexpression (c-Rel is a transcription factor for the IL-12/23 genes),IL-12, or IL-23 expression and sensitivity to apilimod in the testedcell lines.

The expression of IL-12A, IL-12RB1, IL-12RB2, IL-12B, IL-23A and IL-23Rwas analyzed in a diverse group of 75 cancer cell lines (see Table 3).

TABLE 3 Various Cancer cell lines Cell IC50 Number Cancer Model Line(nM) 1 Human Burkitt's lymphoma ST486 25 2 Human Mantle Cell LymphomaJeKo-1 70 3 Human Diffuse Large B Cell SUDHL-4 25 Lymphoma -GCB 4 HumanDiffuse Large B Cell SUDHL-6 80 Lymphoma -GCB 5 Human Burkitt's lymphomaDaudi 200 6 Human histiocytic lymphoma U937 106 7 Human lung carcinomaA549 110 8 Human colorectal cancer HCT116 125 9 Human B-cell lymphoma DB150 10 Human Diffuse Large B Cell WSU-DLCL2 160 Lymphoma -GCB 11 HumanColorectal HCT-15 200 12 Human Colorectal SW480 90 13 Human ColorectalCOLO-205 380 14 Human Colorectal SW620 90 15 Human T-cell leukemiaJurkat 200 16 Human neuroglioma H4 250 17 Human Diffuse Large B CellToledo 270 Lymphoma -GCB 18 Human B cell Non-Hodgkin's Rec-1 300Lymphoma 19 Human Hodgkin's lymphoma KMH-2 181 20 Human Burkitt'slymphoma EB1 174 21 Human Diffuse Large B Cell SUDHL-10 20 Lymphoma -GCB22 Human Burkitt's lymphoma GA-10 382 23 Human Diffuse Large B CellOCI-Ly19 380 Lymphoma -GCB 24 Human Diffuse Large B Cell HT 642 Lymphoma-GCB 25 Human Diffuse Large B Cell Pfeiffer 2,620 Lymphoma -GCB 26 HumanBurkitt's lymphoma Namalwa 600 27 Human Follicular B Cell DOHH-2 700Lymphoma-GCB 28 Human Bladder carcinoma SW780 1000 (GATOR−/−) 29 Humancolorectal cancer MDST8 1000 30 Human Burkitt's lymphoma Raji 10,000 31Human Hodgkin's lymphoma HD-MyZ >1000 32 Human Hodgkin's lymphomaL540 >1000 33 Human Hodgkin's lymphoma HDLM-2 >1000 34 Human Burkitt'slymphoma CA46 >10,000 35 Human Anaplastic Large SUDHL-1 590 CellLymphoma 36 Human lung carcinoma H1734 1500 37 Human colorectal cancerSW1116 1500 38 Human Colorectal COLO-320DM 2,060 39 Human neuroblastomaA172 2000 40 Human lung carcinoma H1693 2000 41 Human lung carcinomaH460 >2000 42 Human lung carcinoma H358 >2000 43 Human pancreatic cancerCAPAN2 >2000 44 Human pancreatic cancer PANC1 >2000 45 Human pancreaticcancer MiaPaCa-2 >2000 46 Human pancreatic cancer AsPC1 >2000 47 Humanprostate cancer DU145 >2000 48 Human acute myelogenous KG-1 >2500leukemia 49 Human prostate cancer LnCap 3000 50 Human T-cell lymphoma HH3,300 51 Human T-cell leukemia MOLT-4 3,300 52 Human prostate cancer22RV1 >5000 53 Human colorectal cancer DLD-1 >5000 54 Human myelogenousleukemia K562 >5000 55 Human colorectal cancer RKO >5000 56 Humanovarian TOV-21G 7000 57 Human prostate cancer PC-3 10,000 58 HumanHodgkin's lymphoma L428 10,000 59 Human plasmacytoma RPMI-8226 >10,00060 Human lung carcinoma NCI-1975 >10,000 61 Human breast cancerCAMA1 >10,000 62 Human neuroblastoma SW1088 >10,000 63 Humanneuroblastoma M0591K >10,000 64 Human neuroblastoma U-118 MG >10,000 65Human neuroblastoma U-87 MG >10,000 66 Human acute monocyticTHP1 >10,000 leukemia 67 Human Diffuse Large B KARPAS-422 >10,000 CellLymphoma -GCB 68 Human Follicular B Cell RL >10,000 Lymphoma 69 HumanMantle Cell Lymphoma GRANTA-519 >10,000 70 Human bronchioalveolarNCI-H1650 >20,000 71 Human bronchioalveolar SW1573 >20,000 72 Humanbronchioalveolar NCI-H1781 >20,000 73 Human bronchioalveolar NCI-H166620,000 74 Human Colorectal LOVO >10,000 75 Human Colorectal HT-29>10,000

Briefly, gene expression data from the CCLE was analyzed for the 75cancer cell lines for which dose response curves against apilimod wereobtained. The expression of each interleukin gene was compared insensitive (IC₅₀ less than 500 nM) and insensitive (IC₅₀ greater than 500nM) lines by unpaired t-test. No statistically significant relationshipwas found with the sole exception of IL-23A (p=0.022). IL-23A has beenpreviously noted to be elevated in apilimod sensitive non small celllung cancer lines, and recombinant IL-23A was noted to increaseproliferation of non small cell lung cancer lines (see Baird et al.2013, supra). Importantly, the statistical significance of IL-23Aexpression in sensitive cancer lines appears to be driven entirely byjust two colon cancer lines. Furthermore IL-23A expression is not astatistically significant predictor of sensitivity in Non-Hodgkin's Bcell lymphoma.

Example 5: Apilimod Inhibits Proliferation of Renal Cancer Cells

The renal cancer cell lines RCC-MF, RCC-ER, RCC-JF, and RCC-JW weregrown in McCoy's 5A medium (Corning), while 786-0, 769-P and RCC-FG2were grown in RPMI-1640 (Corning) and A-704 were grown in MEM (Corning)supplemented with 10% FBS (Sigma Aldrich F2442-500ML, Lot 12D370) andPenicillin/Streptomycin (100×) (CellGro Ref 30-002) and seeded at adensity of 1000, 1200, 1000, 4000, 200, 2000, 1200 and 5000 cells perwell, respectively, into 96 well plates in a final volume of 50 μL.

For single treatment studies, 24 h after seeding, cells were treatedwith apilimod mesylate (referred to in this example as simply ‘apilimod’or LAM-002), sorafenib, pazopanib or sunitinib at a final concentration0.5-10000 nM (3-fold dilutions and a total of 10 dilutions). All drugdilutions were made up as a 2× stock and 50 μL added to appropriatewells. Cells were treated for 120 h before viability was assessed usingCellTiterGlo® (Promega) where the relative luminescence of untreatedcells was set to 100% viability and each drug concentration expressed asa percentage of untreated cells. EC₅₀ values were determined usingGraphPad Prism (GraphPad Software, Inc). Briefly, raw data was logtransformed and then analyzed using nonlinear regression (curve fit)where the data were constrained (bottom=0, top=100).

The results of these single treatment studies are shown in FIGS. 6-13and summarized in the following table.

TABLE 4 Results of single treatment studies in renal cancer cell linesCell line IC50 (nM) 769-P 44 RCC-MF 8 RCC-ER 9 RCC-FG2 32 RCC-JF 60786-0 71 A-704 11 RCC-JW 27

For calculation of synergy between apilimod and pazopanib, or sorafenib,RCC-ER, RCC-FG2, RCC-MF, and 769-P cells were seeded as outlined above.24 h later, cells were treated with apilimod alone (final concentration2-250 nM; 2-fold dilutions and a total of 8 dilutions), with pazopanibalone (final concentration 78.1-10000 nM; 2-fold dilutions and a totalof 8 dilutions) or with sorafenib (final concentration 78.1-10000 nM;2-fold dilutions and a total of 8 dilutions) or the combination of eachconcentration of apilimod with each concentration of pazopanib or ofsorafenib (8×8 matrix). Cells were treated for 120 h before viabilitywas assessed using CellTiterGlo® (Promega) where the relativeluminescence of untreated cells was set to 100% viability and each drugconcentration expressed as a percentage of untreated cells.

FIGS. 14-21 show the results of the synergy studies. Bar graphs in eachfigure (A) show the effect of a single concentration of apilimod, asingle concentration of pazopanib and the effect of the combination ofapilimod and pazopanib (at the single agent concentrations) on cellviability. The ‘expected value’, if the combinatorial effects of the twodrugs were additive, was calculated by using the fraction of viabilityfor apilimod multiplied by fraction of viability for pazopanib, and isshown as the black bar. The CI versus fractional effect graphs (B) ineach figure show the combination index (CI) as defined by Chou et al.(Chou TC, Talalay P. Quantitative analysis of dose-effect relationships:the combined effects of multiple drugs or enzyme inhibitors. Adv EnzymeRegul. 1984; 22:27-55) which was used as a measure of synergy andcalculated using CalcuSyn (version 2.11, Biosoft). The analysis wasconstrained to assess CI values which were within a clinicallyachievable concentration and also where the fraction effect (Fa) wasgreater than 0.75 (i.e., a greater than 75% reduction in cell viabilitywith the combination of drugs). In the CI versus fractional effectgraphs datapoints are denoted by ‘x’ and lines show 95% confidenceinterval, and drug combinations producing CI values>1 are antagonistic,CI=1 are additive, and CI<1 are synergistic. In addition, the CI valueat the ED₅₀, ED₇₅ and ED₉₀ are shown for the combination of apilimod andeither pazopanib or sorafenib. The same methodology was applied for thecombination of apilimod with sorafenib. Tables 6 below summarizes theresults of the synergy studies.

The data presented here demonstrate that apilimod was able to actsynergistically with pazopanib and with sorafenib in the panel of renalcell lines tested.

TABLE 5 CI value at the ED₅₀, ED₇₅ and ED₉₀ for the combination ofapilimod and pazopanib in various cell lines. Cell Line Apilimod+ CI atED₅₀ CI at ED₇₅ CI at ED₉₀ RCC-ER sorafenib 0.77 0.45 0.31 RCC-FG2sorafenib 0.79 0.64 0.51 RCC-MF sorafenib 0.73 0.59 0.48 769-P sorafenib1.22 0.87 0.62 RCC-ER pazopanib 0.56 0.35 0.22 RCC-FG2 pazopanib 0.320.38 0.46 RCC-MF pazopanib 0.58 0.50 0.43 769-P pazopanib 0.57 0.87 1.31

TABLE 6 Summary of synergy studies. RCC-ER RCC-FG2 RCC-MF 769-PPazopanib + Synergistic Synergistic Synergistic Synergistic LAM-002Sorafenib + Synergistic Synergistic Synergistic Synergistic LAM-002

What is claimed is:
 1. A method for treating clear cell renal carcinomain a subject in need thereof, the method comprising administering to thesubject a pharmaceutical composition comprising a therapeuticallyeffective amount of apilimod, or a pharmaceutically acceptable saltthereof.
 2. The method of claim 1, wherein the clear cell renalcarcinoma is refractory to standard treatment or is metastatic.
 3. Themethod of claim 1, wherein the composition is in a form suitable fororal or intravenous administration.
 4. The method of claim 1, whereinthe pharmaceutically acceptable salt is selected from sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, besylate, gentisinate, fumarate, gluconate,glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate. 5.The method of claim 4, wherein the pharmaceutically acceptable salt isselected from chloride, phosphate, lactate, tartrate, maleate, fumarate,methanesulfonate, and pamoate.
 6. The method of claim 1, furthercomprising administering to the subject at least one additional activeagent.
 7. The method of claim 6, wherein the at least one additionalactive agent is a vascular endothelial growth factor (VEGF) inhibitor.8. The method of claim 7, wherein the VEGF inhibitor is selected fromthe group consisting of bevacizumab, sunitinib, pazopanib, axitinib,sorafenib, regorafenib, lenvatinib, motesanib, and vandetanib.
 9. Themethod of claim 8, wherein the VEGF inhibitor is selected from pazopanibor sorafenib, or a combination thereof.
 10. The method of claim 6,wherein the at least one additional active agent is a PD-1/PDL-1 pathwayinhibitor.
 11. The method of claim 10, wherein the at least oneadditional active agent is selected from pembrolizumab, avelumab,atezolizumab (MPDL3280A), nivolumab (BMS-936558), pidilizumab (MK-3475),MSB0010718C, and MEDI4736.